Bottom Line:
Adeno-associated viral (AAV) vectors show great promise for gene therapy because of their excellent safety profile; however, development of robust dose-determining assays for AAV has presented a significant challenge.With the ultimate goal of future harmonization and standardization of AAV dose determination assays, we systematically analyzed the influence of key variables, including sample preparation procedure, the choice of primers, and real-time quantitative PCR (qPCR) target sequences and calibration DNA conformation on the qPCR quantitation of AAV products.Our results emphasize the importance of designing qPCR primers and conducting sample preparation and demonstrate the need for extensive characterization, vigorous control, and use of reference materials in clinical dose determination.

ABSTRACTAdeno-associated viral (AAV) vectors show great promise for gene therapy because of their excellent safety profile; however, development of robust dose-determining assays for AAV has presented a significant challenge. With the ultimate goal of future harmonization and standardization of AAV dose determination assays, we systematically analyzed the influence of key variables, including sample preparation procedure, the choice of primers, and real-time quantitative PCR (qPCR) target sequences and calibration DNA conformation on the qPCR quantitation of AAV products. Our results emphasize the importance of designing qPCR primers and conducting sample preparation and demonstrate the need for extensive characterization, vigorous control, and use of reference materials in clinical dose determination.

f6: Comparison of AAV genome titers in samples prepared by method A (hatched columns) or method B (gray columns). Vector titers (VG/ml) were calculated on the basis of circular plasmid [(A), (C), and (E)] or linear plasmid [(B), (D), and (F)], using ITR primers (A and B), GFP primers (C and D), or CMV primers (E and F). Data shown represent mean data of nine PCRs of the same sample extracted in triplicates and then qPCR in triplicate (n=9). Asterisks indicate statistical significance observed between the two methods, where *p<0.05, **p<0.01, ***p<0.001.

Mentions:
Four batches of AAV8 vectors, including two high-titer clinical-grade batches (Fig. 6, batches 1 and 2) and two batches of low-titer AAV8 samples (batches 3 and 4; Fig. 6) were prepared by method A (hatched columns; Fig. 6) or method B (gray columns; Fig. 6) and were then qPCR quantified using target sequence ITR (Fig. 6A), GFP (Fig. 6B), or CMV (Fig. 6C). Figure 6 shows that when a statistically significant method variation (p<0.05, indicated by asterisks) was observed, for example, in high-titer clinical-grade samples (batches 1 and 2), the samples treated by method B (Fig. 6A and B, gray columns) showed significantly higher titers than the samples treated by method A, indicating potential interference of capsid proteins in vector quantitation and the importance of sample purity in sample quantitation. It is possible that the method B-associated high titer might partially be due to the increased background detection during the multistep method B (Fig. 6). It is also noteworthy that significant method variation was less apparent in low-titer samples (batches 3 and 4) (<108 VG/ml). This may be due to the loss of the low concentrations of vector DNA during column purification. A typical clinical batch of AAV8 product has a titer exceeding 1012 VG/ml; therefore, on the basis of our results, it is important to include an additional purification step in sample preparation for qPCR quantitation, particularly when using generic ITR qPCR quantitation. Our study also shows that method variations were qPCR target sequence dependent, as no significant variation was observed in qPCR targeting the CMV sequence (Fig. 6E and F), which is in contrast to qPCR targeting ITR (Fig. 6A and B) and GFP (Fig. 6C and D) sequences.

f6: Comparison of AAV genome titers in samples prepared by method A (hatched columns) or method B (gray columns). Vector titers (VG/ml) were calculated on the basis of circular plasmid [(A), (C), and (E)] or linear plasmid [(B), (D), and (F)], using ITR primers (A and B), GFP primers (C and D), or CMV primers (E and F). Data shown represent mean data of nine PCRs of the same sample extracted in triplicates and then qPCR in triplicate (n=9). Asterisks indicate statistical significance observed between the two methods, where *p<0.05, **p<0.01, ***p<0.001.

Mentions:
Four batches of AAV8 vectors, including two high-titer clinical-grade batches (Fig. 6, batches 1 and 2) and two batches of low-titer AAV8 samples (batches 3 and 4; Fig. 6) were prepared by method A (hatched columns; Fig. 6) or method B (gray columns; Fig. 6) and were then qPCR quantified using target sequence ITR (Fig. 6A), GFP (Fig. 6B), or CMV (Fig. 6C). Figure 6 shows that when a statistically significant method variation (p<0.05, indicated by asterisks) was observed, for example, in high-titer clinical-grade samples (batches 1 and 2), the samples treated by method B (Fig. 6A and B, gray columns) showed significantly higher titers than the samples treated by method A, indicating potential interference of capsid proteins in vector quantitation and the importance of sample purity in sample quantitation. It is possible that the method B-associated high titer might partially be due to the increased background detection during the multistep method B (Fig. 6). It is also noteworthy that significant method variation was less apparent in low-titer samples (batches 3 and 4) (<108 VG/ml). This may be due to the loss of the low concentrations of vector DNA during column purification. A typical clinical batch of AAV8 product has a titer exceeding 1012 VG/ml; therefore, on the basis of our results, it is important to include an additional purification step in sample preparation for qPCR quantitation, particularly when using generic ITR qPCR quantitation. Our study also shows that method variations were qPCR target sequence dependent, as no significant variation was observed in qPCR targeting the CMV sequence (Fig. 6E and F), which is in contrast to qPCR targeting ITR (Fig. 6A and B) and GFP (Fig. 6C and D) sequences.

Bottom Line:
Adeno-associated viral (AAV) vectors show great promise for gene therapy because of their excellent safety profile; however, development of robust dose-determining assays for AAV has presented a significant challenge.With the ultimate goal of future harmonization and standardization of AAV dose determination assays, we systematically analyzed the influence of key variables, including sample preparation procedure, the choice of primers, and real-time quantitative PCR (qPCR) target sequences and calibration DNA conformation on the qPCR quantitation of AAV products.Our results emphasize the importance of designing qPCR primers and conducting sample preparation and demonstrate the need for extensive characterization, vigorous control, and use of reference materials in clinical dose determination.

ABSTRACTAdeno-associated viral (AAV) vectors show great promise for gene therapy because of their excellent safety profile; however, development of robust dose-determining assays for AAV has presented a significant challenge. With the ultimate goal of future harmonization and standardization of AAV dose determination assays, we systematically analyzed the influence of key variables, including sample preparation procedure, the choice of primers, and real-time quantitative PCR (qPCR) target sequences and calibration DNA conformation on the qPCR quantitation of AAV products. Our results emphasize the importance of designing qPCR primers and conducting sample preparation and demonstrate the need for extensive characterization, vigorous control, and use of reference materials in clinical dose determination.